Project Summary/Abstract Leukemic relapse remains the major obstacle to successful allogeneic stem cell transplantation (allo-SCT). Here, donor lymphocyte infusion (DLI) seeks to repair the underlying graft-versus-leukemia (GvL) effect that drives allo-SCT efficacy. However the mechanisms driving clinical GvL outcomes remain poorly understood. To address this challenge, Dr. Bachireddy has performed deep molecular phenotyping of both leukemia and immune cells within the tumor microenvironment (TME) across multiple timepoints during DLI response and resistance. He previously showed reversal of T cell exhaustion, a unique form of T cell dysfunction, during DLI response. Using high-throughput single cell RNA sequencing (scRNA-seq), he has now identified 2 main subtypes of exhausted T cells: terminally exhausted (TE) cells, enriched in pre-DLI responding TMEs, and progenitor exhausted (PE) cells, expanding consistently in responders post-DLI. While these subsets are defined in murine models of T cell exhaustion that motivated inhibitors of the PD-1 pathway, their relevance to human leukemia, roles in immunotherapeutic response, and relationship to oncogenic pathways have not been previously elucidated. Thus, he will test the hypothesis that specific gene regulatory networks (GRNs) (1) define the heterogeneity within and between TE and PE subsets and, moreover, (2) are influenced by leukemic-derived mutations and gene expression states. In Aim 1, he will determine the regulation and function of TE and PE subsets by, first, integrating scRNA-seq data with chromatin accessibility profiles to identify GRNs for each subset and then functionally evaluating the relative anti-leukemic efficacy of each subset (and each GRN) in vivo. In Aim 2, he will identify leukemic-derived molecular profiles that associate with DLI outcome and shape T/PE T cell subsets. Leukemic genomic and scRNA-seq data will be jointly analyzed to perform in silico identification of candidate leukemic drivers of DLI outcome and T/PE T cell subsets, which will, in turn, be functionally evaluated in vivo. These results will identify GRNs that govern, and oncogenic pathways that shape, TE and PE T cells, revealing novel targets for modulating cancer immunity and uncovering the molecular circuitry underpinning leukemic-immune interactions in the post-transplant TME. Dr. Bachireddy has outlined a five-year career development plan to meet his goal of becoming an independent investigator focused on immunotherapy in hematologic malignancies. He has assembled an Advisory Committee of internationally recognized experts to provide scientific/career mentorship and enlisted collaborators who are experts in computational biology, systems immunology, and functional genomics to provide experimental advice and specific training in the field. The Dana-Farber Cancer Institute and Broad Institute of MIT and Harvard are the ideal environments for completion of his scientific and career goals, given their outstanding research communities and substantial records for training independent physician-scientists.